SK285637B6 - Propellant-free nebulizer containing an aerosol preparation, process for preparing propellant-free aerosols and use of nebulizer - Google Patents

Abstract

Described is propellant-free nebulizer containing an aerosol preparation in the form of a solution and a process for preparing aerosols for administration by inhalation of aerosol preparation. In propellant-free nebulizer a therapeutically active quantity of an aerosol preparation is sprayed, being measured in a measuring chamber and under high pressure of between 10 MPa and 50 Mpa through at least one nozzle with a hydraulic diameter of 1 to 12 micrometers droplets with an average particle size of less than 10 micrometers within a period of between one and two seconds.

Description

The invention relates to a method of generating aerosols for administration of proteins and other biologically active macromolecules by inhalation, as well as aqueous formulations for generating such aerosols, in particular the invention relates to aqueous formulations of highly concentrated insulin solutions for inhalation administration for the treatment of diabetes.

BACKGROUND OF THE INVENTION

The use of medicaments in the form of inhalable aerosols has been known for a long time. Such aerosols serve not only for the treatment of respiratory diseases such as asthma; they are also used when the lungs or nasal mucous membranes are to serve as a resorptive organ. In this way, it is often possible to produce such high levels of the active substance in the blood that diseases in other areas of the body can be treated. Inhalable aerosols may also serve as vaccines.

In practice, several methods are used to generate aerosols. Either the suspensions or solutions of the active substances are sprayed with propellants, or the active substances in the form of micronized powders are vortexed in the inhaled air, or finally the aqueous solutions are sprayed with the aid of nebulizers.

However, for molecules with complex structures, such as interferons, spraying of aqueous solutions can easily lead to a disruptive reduction in the activity of the active ingredient, possibly due to shear forces and heating. For example, the formation of less active protein aggregates is believed to play a role in this process. A. Y. Ip et al. described in their article "Stability of Recombinant Consensus Interferon to Air-Jet and Ultrasonic Nebulization", J. Pharm. Sci. 84, 1210-1214, 1995, examples of the formation of interferon aggregates by ultrasonic or nozzle spraying with the occurring loss of interferon biological activity. Although the damage to the biomolecule (biologically active macromolecule) is incomplete, the reduction in activity described herein is important because it causes increased consumption, or expensive biomolecule, and dosing of the active drug per stroke becomes inaccurate. This reduction in the activity of complex structure molecules during aerosol formation is not limited to interferons, but also occurs to a lesser or greater extent when other proteins are aerosolized (see, e.g., Niven et al., Pharm. Res. 12, 53-59, 1995). abiomolekúl.

In addition to the technical preparation of an aerosol containing a biomolecule, a second step is necessary to absorb the biomolecules in the lungs. The adult's lungs offer a large absorption area, but it also presents several barriers to the pulmonary absorption of biomolecules. After inhalation through the nose or mouth, the air with the aerosol it carries enters the trachea and then through the ever-smaller bronchi and bronchial tubes into the alveoli. Alveoli have a much larger area than the trachea, bronchi, and bronchial tubes together. They are the main absorption zone not only for oxygen but also for biologically active macromolecules. In order to get out of the air into the bloodstream, the molecules must cross the alveolar epithelium, capillary endothelium and lymph containing the interstitial space between the two cell layers. This can be accomplished by active or passive transport processes. The cells in the two cell layers lie close together so that most large biological macromolecules (such as proteins) will overcome this barrier more slowly than smaller molecules. The passage through the alveolar epithelium and capillary endothelium is in competition with other biological processes that lead to biomolecular damage. Bronchoalveolar fluid contains exoproteases (see, for example, Wall DA and Lanutti AT, "High levels of excretidase activity present in rat and canine bronchoalveolar lavage fluid" (High levels of exopeptidase activity are present in bronchoalveolar lavage fluid in rats and dogs), Int. J Pharm. 97: 171-181 (1993). It also contains macrophages that eliminate inhaled protein particles through phagocytosis. These macrophages migrate to the base of the bronchial tree, from where they come from the lungs through a mucosal cleansing mechanism. They can then migrate to the lymphatic duct. Furthermore, the acrosolized protein can affect the physiology of macrophages, for example interferons can activate alveolar macrophages. Activated macrophage migration is another mechanism for enhancing the systemic effect of the inhaled protein. The complexity of this process means that the results of experiments with aerosols with one type of protein can be transmitted to another type of protein only to a limited extent. Small differences between interferons may, for example strong influence on their susceptibility to degradation mechanisms in the lungs (see Bocci et al., "Pulmonary Catabolism of Interferons: alveolar absorption of ¹²⁵ I labeled human interferon alpha is accompanied by partial loss of biological activity" (Pulmonary interferon catabolism: the alveolar absorption of ¹²⁵ I-labeled human interferon alpha is associated with a partial loss of biological activity ', Antiviral Research 4, 211-220, 1984).

Proteins and other biological macromolecules may in principle be sprayable, but this spraying generally occurs with a loss of activity. It is an object of the present invention to provide a method of generating inhalable aerosols by which biologically active macromolecules, in particular proteins, can be atomized without significant loss of activity.

A new generation of propellant-free atomisers is described in U.S. Patent 5,497,944, the contents of which are hereby incorporated by reference. A particular advantage of the atomizers described therein is that propellants, in particular fluorine-chlorine-hydrocarbons, are not used.

A further developed embodiment of the atomizers described therein is disclosed in PCT / EP96 / 04351 = WO 97/12687. In the context of the present invention, reference is made explicitly to Figure 6 (Respimat®) described therein, as well as to the parts of the specification described therein. The nebuliser described herein may advantageously be used to generate the inhalable aerosols of the present invention of biologically active macromolecules. In particular, the nebuliser described can be used for inhaled administration of insulin. Due to its convenient size, the patient can always carry the device with him. In the nebulizer described therein, solutions of a defined volume (preferably about 15 microlitres) containing the active ingredient are sprayed over small nozzles using high pressures to produce inhalable aerosols with a mean particle size of between 3 and 10 microns. For the inhaled administration of insulin, nebulizers that can spray between 10 and 50 microliters of a disposable aerosol formulation onto inhalable droplets are suitable.

Of particular importance for the generation of aerosols according to the invention is the use of the sprayer described in said patent, respectively. Patent application for spraying active substance-containing solutions containing proteins or other biologically active macromolecules without propellant.

The easy-to-use dispenser disclosed therein (aerosol dispenser, size about 10 cm) consists essentially of an upper housing part, a pump housing, a nozzle, a locking tensioner, a spring housing, a spring and a reservoir, further comprising:

a pump housing which is fixed in the upper housing part and which carries a nozzle body with a nozzle or a nozzle at one end thereof; nozzle assembly,

- hollow piston with valve body,

- a driven flange in which the hollow piston is attached and which is located in the upper housing part,

- locking tensioner located in the upper housing part,

- a spring housing with a spring therein which is rotatably mounted on the upper housing part by means of a rotatable bearing,

a lower housing part which is mounted in the axial direction on the spring housing.

The hollow piston with valve body WO 97/12687 corresponds to one of the disclosed devices. It partially protrudes into the cylinder of the pump housing and is arranged in the cylinder axially displaceably. Referring in particular to FIG. 1 to 4 - in particular in FIG. 3 - and the corresponding parts of the description. The hollow piston with the valve body exerts a pressure on the fluid, i.e. a measured amount of the active substance solution, from 5 to 60 MPa (about 50 to 600 bar), preferably 10 to 60 MPa (about 100 to 600 bar) on its high-pressure side. .

The valve body is preferably located at the end of the hollow cylinder that faces the nozzle body.

The nozzle in the nozzle body is preferably microstructured, i. j. is made by micro technology. Microstructured nozzle bodies are disclosed, for example, in WO-94/07607; We hereby refer to the contents of this file.

The nozzle body consists, for example, of two glass and / or silicon plates fixed to one another, of which at least one plate has one or more microstructured channels connecting the inlet side of the nozzle to the outlet side of the nozzle. At least one round or non-round orifice having a diameter of less than or equal to 10 µm is located at the outlet side of the nozzle.

The directions of jet streams in the nozzle body may extend parallel to each other or at an angle to each other. In a nozzle body with at least two nozzle openings on the outlet side, the flow directions can be at an angle of 20 to 160 degrees to each other, preferably at an angle of 60 to 150 degrees. The flow directions meet around the nozzle openings.

The locking tensioner comprises a spring, preferably a cylindrical helical compression spring, as a reservoir of mechanical energy. The spring acts on the driven flange as a step element, the movement of which is determined by the position of the locking member. The path of the driven flange is precisely limited by the upper and lower stops. The spring is preferably tensioned by a transmission transmitting force, for example a helical sliding transmission, by an external torque which develops when the upper housing part is rotated against the spring housing in the lower housing part. In this case, the upper housing part and the driven flange comprise a single or multi-stage wedge gear.

The locking member with the engaging locking surfaces is annularly arranged around the driven flange. It consists, for example, of a radially elastically deformable grille of plastic or metal. The ring is arranged perpendicular to the atomizer axis. When the spring is tensioned, the locking surfaces of the locking member move into the path of the driven flange and prevent the spring from loosening. The locking member is released using the button. The release button is coupled or coupled to the locking member. To release the locking tensioner, the release button is displaced parallel to the plane of the barrel, preferably into the sprayer; the deformable ring is deformed in its plane. The construction details of the locking tensioner are described in WO 97/20590.

The lower housing part is moved in the axial direction through the spring housing and covers the bearing, the spindle drive and the fluid reservoir.

When actuating the sprayer, the upper housing part is rotated against the lower housing part, the lower housing part pulling the spring housing therewith. In this case, the spring is compressed and tensioned by means of a helical sliding gear and the locking device automatically engages. The angle of rotation is preferably an integer fraction of 360 degrees, for example 180 degrees. Simultaneously with the spring tension, the driven part in the upper housing part is displaced by the prescribed path, the hollow piston is retracted inside the cylinder in the pump housing, so that a part of the liquid is sucked from the reservoir into the high pressure space before the nozzle.

Optionally, a plurality of replaceable reservoirs containing the fluid to be atomized may be inserted and used in the sprayer. The reservoir comprises an aqueous aerosol formulation according to the present invention.

The spraying process is started by lightly pressing the release button. In this case, the locking device makes way for the driven part. The tensioned spring slides the piston into the cylinder of the pump housing. The fluid emerges from the atomizer nozzle in a sprayed form.

Further construction details are disclosed in PCT applications WO 97/12683 and WO 97/20590, the contents of which are hereby incorporated by reference.

The components of the sprayer (aerosol sprayer) are of a material which is appropriately suitable for their function. The spray case and, if their function permits, other parts are preferably made of plastic, for example by injection molding. Physiologically harmless materials are used for medical purposes.

For example, the nebulizer disclosed in WO 97/12687 is used to generate propellant-free medical aerosols. This can produce an inhalable aerosol with a mean droplet size of about 5 µm.

In FIG. 4a / b. which are identical to FIG. 6a / b of WO 97/12687, there is shown an aerosol dispenser (Respimat®) with which the aqueous aerosol formulations of the present invention can be advantageously inhaled.

Fig. 4a shows a longitudinal section through the atomiser with the spring tensioned; FIG. 4b shows a longitudinal section through the atomiser with the spring released.

The upper part 51 comprises a pump housing 52, at the end of which the sprayer nozzle holder 53 is located. A nozzle body 54 and a filter 55 are located in the holder. The hollow piston 57, which is mounted in the driven flange 56 of the locking tensioner, partially extends into the cylinder of the pump housing. The hollow piston carries a valve body 58 at its end. The hollow piston is sealed by means of a gasket 59. Inside the upper housing part there is a stop 60 to which the driven flange abuts when the spring is released. At the driven flange there is a stop 61 to which the driven flange abuts when the spring is tensioned. After the spring is tensioned, the locking member 62 is inserted between the stop 61 and the support 63 in the upper housing part. The release button 64 is in communication with the locking member. The upper housing part terminates in the mouthpiece 65 and is closed by a tunable protective cap 66.

The spring housing 67 with the spring-printing 68 is rotatably mounted on the upper housing part by means of the snap-in thumb 69 and the pivot bearing. The lower housing part 70 is inserted through the spring housing. Inside the spring housing there is a replaceable fluid reservoir 71 for the fluid 72 to be atomized. The reservoir is closed by a plug 73, through which the hollow piston protrudes into the reservoir and, at its end, is immersed in the liquid (active substance solution stock).

A mechanical counter spindle 74 is disposed on the housing surface of the spring housing. At the end of the spindle which faces the upper housing part there is a driven pinion 75. A slider 76 is disposed on the spindle.

The disclosed aerosol dispenser is suitable for spraying the aerosol formulations of the present invention into an aerosol suitable for inhalation.

The efficiency of the nebulizer can be tested in an in vitro system in which the protein solution is sprayed and the mist is trapped in a so-called "trap" (see Fig. 1). The activity of the protein in the aerosol container a is compared with the activity in the entrapped fluid b, for example by means of an immune assay or a protein bioassay assay. This experiment makes it possible to assess the degree of protein damage by spraying. The second parameter of aerosol quality is the so-called inhalable fraction, which is defined here as the proportion of mist droplets with a median median diameter (MMAD) of less than 5.8 µm. The inhalable fraction can be measured using an "Andersen impactor". For good protein uptake, it is important not only to achieve nebulization without significant loss of activity, but to generate an aerosol with a good (about 60%) inhalable fraction. Aerosols with an MMAD of less than 5.8 µm are significantly more suitable for achieving alveoli, where their chances of being absorbed are significantly greater. The efficacy of the nebulizer can also be tested in an in vivo system, in which factors such as lung protease sensitivity play a role. As an example of an in vivo test system, a dog containing a protein may be administered through a tracheal tube. Blood samples are taken at appropriate time intervals and then plasma protein levels are measured by immunological or biological methods.

Suitable nebulizers are described in the aforementioned U.S. Pat. No. 5,497,944 and WO 97/12687, in particular as in FIG. 6a / b (now 4a / b). A preferred nozzle assembly for spraying the aqueous aerosol formulations of the biologically active macromolecules of the present invention is shown in FIG. 8 of said U.S. Pat.

Surprisingly, it has been found that the described propellant-free aerosol dispenser which atomises a predetermined amount - for example 15 microlitres - of an aerosol formulation under high pressure between 10 MPa and 50 MPa (100 and 500 bar) through at least one nozzle with a hydraulic diameter of 1 to 12 microns so that inhalable droplets with a mean particle size of less than 10 microns are formed, it is well suited for spraying liquid aerosol formulations of proteins and other macromolecules as it can spray a wide variety of proteins without significant loss of activity. A nozzle assembly as shown in FIG. 8 of said U.S. Pat.

Particularly surprising is the ability of aerosol dispensers with this type of construction to spray interferons which otherwise can only be sprayed with a significant loss of activity. Furthermore, the particularly high activity of interferon Omega after spraying with this apparatus is surprising, not only in in vitro but also in in vivo experiments.

WO 95/03034 also deals with the stabilization of aqueous protein aerosols. However, it solves this problem through organic polar compounds or surfactants.

A further advantage of the claimed method is its surprising property that it can also spray highly concentrated solutions of biologically active macro molecules without significant loss of activity.

The use of highly concentrated solutions allows the use of a device that is small enough to be worn comfortably in a jacket pocket or handbag. The aerosol dispenser disclosed in FIG. 4, fulfills these requirements and allows spraying of highly concentrated solutions of biologically active molecules.

SUMMARY OF THE INVENTION

The present invention is defined by the claims and relates to a propellant-free nebulizer comprising an aerosol formulation, its use for the preparation of aerosols, as well as a method of producing propellant-free aerosols.

For example, such devices are particularly suited to enable diabetics to inhale insulin self-treatment. Highly concentrated solutions with a concentration of from 20 to 90 mg / ml insulin are preferred, more preferred are solutions with 30 to 60 mg / ml insulin, and particularly preferred are insulin solutions with 33 to 40 mg / ml insulin. Depending on the size of the aerosol dispenser container available, solutions containing insulin at a concentration of greater than 25 mg / ml, preferably greater than 30 mg / ml, are suitable for inhalation of a therapeutically effective amount with a handheld device such as the device described. insulin. The inhaled administration of insulin allows for the rapid onset of action of the active ingredient so that, for example, the patient can inject the necessary amount of it shortly before meals. For example, the small size of the Respimat® allows the patient to carry the device with him at all times.

Respimat® (Fig. 6 in WO 97/12687) has a constant volume dosing chamber that allows the patient to determine the required insulin dose for himself by the number of strokes (pufTs) and to inhale. In addition to the number of strokes, the insulin dosage is determined by the concentration of the insulin solution in the reservoir 72. It may, for example, be between 25 and 90 mg / ml, more concentrated solutions of about 30 mg / ml and more are preferred.

Methods for preparing highly concentrated insulin solutions are described, for example, in WO-applications 83/00288 (PCT / DK82 / 00068) and 83/03054 (PCT / DK83 / 00024), the contents of which are hereby incorporated by reference.

The aerosol formulations of the present invention containing insulin that are administered by the described device should not exceed a dynamic viscosity of 1600 x ^{10 6} Pa.s so that the inhalable fraction of mist formed does not fall below an acceptable value. Insulin solutions which exhibit a cut-off viscosity of up to 1200 x ^{10 6} Pa.s (Pascal-second) are preferred. If necessary, solvent mixtures may be used instead of water to reduce the viscosity of the drug solution. This can be done, for example, by adding ethanol. The proportion of ethanol in the aqueous formulation may be, for example, 50%, preferably 30%.

The aerosol formulation preferably has a viscosity of up to 1600 x ^{10 6} Pa.s, with an area of 900 to 1100 x ^{10 6} Pa.s being particularly preferred.

Also preferred are aerosol formulations whose aqueous solutions have a viscosity between 900 and 1600 x ^{10 6} Pa · s, aqueous solutions with a viscosity in the range from 950 to 1300 x ^{10 6} Pa · s are particularly preferred.

Another object of the present invention is to provide a suitable aerosol formulation suitable for use in the claimed method.

The invention also relates to an aerosol formulation in the form of a solution with water or with a water / ethanol mixture as a solvent, which contains, as active ingredient in a concentration of 25 to 100 mg / ml, biologically active macromolecules, in particular a protein or peptide. from the group: antisense oligonucleotides, orexins, erythropoietin, tumomecrotic factor alpha, tumomecrotic factor beta, granulocyte colony stimulating factor - G-CSF, granulocyte macrophage colony stimulating factor - GM-CSF, annexin, calcitonin, leptin, parathormone, parathormone, parathormone such as interleukin 2, interleukin 10, interleukin 12, intercellular adhesion molecule-soluble ICAM; somatostatin, somatotropin, Tissue plasminogen activator - tPA; TNK-tPA, tumor-associated antigens, for example peptide, protein or as DNA, peptide bradykinin antagonists, urodilatin, growth hormone releasing hormone - GHRH, corticotropin releasing factor - CRF, Ε ΑΡ II, heparin, soluble interleukin receptors such as sIL-1 receptor, vaccines such as hepatitis vaccine or measles vaccine, antisense polynucleotides, transcription factors, to produce inhalable aerosols via a propellant-free nebulizer.

Surprisingly, it has been found that the highly viscous macromolecule solutions can also be sprayed onto inhalable droplets of a suitable droplet size by the method of the invention. This allows the administration of larger amounts of the active ingredient for single use and thereby increases the therapeutic efficacy of the macromolecules in inhalation therapy.

Aqueous aerosol formulations containing macromolecules (for example albumin) up to a viscosity of 1600 x ^{10 6} Pa.s (measured at 25 ° C) can be used in the method of the invention. The viscosity of 1500.10 ^'6 Pa has not found inhalable proportion of 32%.

Preferred are more viscous solutions of macromolecules having a viscosity of up to 1100 x ^{10 6} Pa.s. In such solutions, an inhalable proportion of droplets containing the active ingredient of about 60% is obtained. Said cut-off viscosities were determined by an Ostwald viscometer by a method known in the literature. By comparison, the viscosity of water is 894 x ^{10 6} Pa · s (measured at 25 ° C).

In order to illustrate the advantages of the method of the invention, in vitro and in vivo experiments with a solution of interferon Omega are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1 is a Respimat® aerosol dispenser.

In FIG. 2 shows measurements of Omega interferon (a) with an immune test (ELISA) or (b) with a bioassay (antiviral activity) of Dog # 2. 95-1467.

In FIG. 3 is a graph of neopterin release in plasma following aerosol administration; or i. in. Omega Dog no. 95-1467.

Fig. Fig. 4a shows a longitudinal section through the atomizer (Respimat®) with the spring tensioned; 4b shows a longitudinal section through the atomizer (Respimat®) with the spring released.

In FIG. 5 is a graph depicting the amount of blood glucose after administration of insulin by Respimat-dog no. 951,479th

DETAILED DESCRIPTION OF THE INVENTION

In vitro experiments with Respimat® and Omega interferon

These experiments were performed in the Respimat® apparatus shown in FIG. 1, which consists of a reservoir a for liquid, a trap c, a sintered glass frit e and a connecting piece d. The reservoir and the Respimat instrument were filled with a solution of 5 mg / ml Omega interferon (formulated in mM trisodium citrate, 150 mM NaCl, pH 5.5). The apparatus was activated and a volume of about 12.9 µl (one stroke) was sprayed in an air flow of 28 L / min. The sprayed solution b was trapped (Fig. 1). Interferon Omega was determined in ELISA and biologically, by inhibiting damage to A549 cells infected with encephalomyocarditis virus in solution in the reservoir and trapped solution. Immunological determination of interferon is relatively simple. Published experiments with atomized proteins are in many cases limited to immunological measurements. However, additional biological measurements are very important as they are a particularly sensitive and therapeutically important method of quantifying protein damage. They do not always give the same results as physicochemical or immunological methods, because the molecule can lose biological ability without altering its binding to antibodies.

In three experiments, based on the starting solution, 84%, T1% and 98% of the immunologically identifiable interferon in solution b were recovered from the trap. Biological measurements with the same solutions yielded 54%, 47% and 81% of the recovered biologically identifiable interferon in the trap solution. This very high proportion shows that spraying with a Respimat device disrupts only a small part of interferon activity. The mist from the Respimat instrument, as described, was also introduced into the Andersen impactor via an air flow (28 L / min). The fraction of particles smaller than 5.8 µm ("inhalable fraction") was measured. The inhalable proportion was 70% (immunological measurements). Proteins such as interferons are often formulated with human serum albumin to provide additional protection for sensitive interferons. One formulation as above, but with additional human serum albumin (0.5%) was also tested. In three experiments, also based on the starting solution, 83%, 83% and 79% of the immunologically identifiable interferon in solution b were recovered from the trap. Biological measurements with the same solutions yielded 60%, 54% and 66% of biologically active interferon in the trap solution. The inhalable proportion (immunoassays) was 67%. In another batch, a concentrated Omega interferon solution of 53 mg / ml was filled into a Respimat reservoir and then sprayed. In four experiments, based on the starting solution, 100%, 60%, 68% and 72% of the immunologically identifiable interferon was again detected in solution b from the trap. Biological measurements with the same solutions yielded 95%, 98%, 61% and 83% of the recovered, biologically identifiable interferon in the trap solution. These high re-findings show that concentrated protein solutions can also be sprayed with the Respimat apparatus without causing excessive loss of interferon activity.

In vivo experiments with Respimat® and interferon Omega

Interferon Omega was administered by inhalation and intravenous injection to the same dog. Blood interferon levels were measured immunologically and biologically at various times. In addition, blood levels of neopterin were measured. Neopterin is a marker of immune activation; it is released by macrophages after irritation by interferon (see Fuchs et al., "Neopterin, biochemistry and clinical use as a marker for cellular immune reactions"), Int. Allergy Appl. Immunol., 101, 1-6, 1993). Measurement of neopterin levels serves to quantify interferon efficacy.

Dog administration of interferon was performed under pentobarbital anesthesia following prior administration of basal sedatives. The animal was intubated and artificial respiration was introduced (volume controlled breathing: minute volume of 4 l / min, frequency of 10 strokes / min). A total of 20 strokes were received from the Respimat. Each stroke was applied at the beginning of the breath. After the inhalation phase, there was a 5-second break before exhalation. Before the next administration of Omega interferon, the animal was allowed two breathing cycles without affecting it. Blood for serum and heparin plasma was collected before and at various times up to 14 days after interferon administration. Interferon Omega in heparin plasma was determined immunologically by ELISA and biologically inhibited damage to A549 cells infected with encephalomyocarditis virus. Serum neopterin was determined immunologically. Fig. 2 shows immunological (FIG. 2a) and biologically (FIG. 2b) measured levels of Omega interferon after a 20-stroke dose of Omega interferon from the Respimat apparatus. Surprisingly, a very high serum level of neopterin was measured after inhalation. In an in vitro experiment, the amount of solution plotted after a single stroke of the Respimat was an average of 12.8 mg / stroke. Therefore, with a solution of 5 mg / ml, it is expected that about 1.28 mg of interferon will be delivered with 20 strokes of Respimat. Neopterin measurements after administration of this amount yielded significantly higher and longer lasting levels than neopterin measurements after administration of 0.32 mg interferon i. in. This result is illustrated in FIG. 3. High levels of neopterin serve as evidence that the administration of interferon with Respimat can lead to good biological efficacy.

The benefits of the Respimat spraying of biologically active macromolecules are not limited to interferons, as shown in the second example.

In vitro experiments with Respimat® and manganese superoxide dismutase

The test substance nebulizer and associated traps were as shown in FIG. L In this experiment, the reservoir and the Respimat instrument were filled with 3.3 mg / ml manganese superoxide dismutase (MnSOD) in phosphate buffered saline (PBS). The instrument was activated and a volume of about 13 µl (one stroke) was sprayed in an air flow of 28 l / min. The exact spray amount was determined gravimetrically (measurements in three consecutive experiments: 12.8, 13.7 and 14.3 mg). The sprayed solution was trapped in trap b. The content of this trap was 20 ml PBS. Additionally, 2 ml of 5% bovine serum albumin was added to stabilize the trapped proteins. MnSOD was determined in stock solution and trapped solution by immunoassay by ELISA and enzymatically reducing the amount of superoxide following the xanthine / xanthine oxidase reaction. In three experiments, 78%, 89% and 83% of immunologically identifiable MnSOD spray solution were measured in trap solution b. No measurable loss of enzymatic activity was observed after spraying. The inhalable proportion (immunoassays) was 61%.

In the following example, we describe the preparation of an aerosol formulation according to the invention which contains insulin as the active ingredient.

Preparation of the insulin solution and filling of the nebuliser

175 mg crystalline insulin (sodium salt) from cattle (corresponding to 4462.6 IU according to the manufacturer's data) was dissolved in 3.5 ml of sterile, purified water (Seralpur®-water). Then 8.5 μΐ m-krczol (corresponding to 8.65 mg) and 7.53 mg phenol dissolved in 100 μΐ sterile, purified water were added under gentle stirring. To this solution was added 365 μΐ of a 5 mg / ml ZnCl ₂ solution (corresponding to 0.5% zinc by weight based on the amount of insulin used) and the pH was adjusted to 7.4 with 0.2 N NaOH. The volume of the mixture was made up to 5 ml with sterile, purified water and filtered through a sterile Milipore filter (0.22 µm pore size). 4.5 ml of this aerosol formulation was transferred to a reservoir (72 in Fig. 4) of an aerosol dispenser (Respimat). The reservoir was capped and inserted into the device.

The aerosol formulation so produced has a concentration of about 35 mg / ml insulin, the viscosity of this solution being about 1020 x ^{10 6} Pa.s.

In vivo experiment with Respimat® and highly concentrated insulin solution

Insulin administration in the dog was performed under pentobarbital anesthesia following prior administration of basal sedatives. The animal was intubated and artificial respiration was introduced as before. A total of 6 strokes of insulin solution from the Respimat device were administered. Each stroke was applied at the beginning of the breath. There was a break of 5 seconds between the inhalation phase and the exhalation phase. Before the next insulin injection, the animal was allowed two breathing cycles without affecting it. Blood was collected 1 hour prior to administration, concomitantly and at different times over 8 hours. Blood glucose was measured in fresh blood by the method of Trasch, Koller and Tritschler (Klein. Chem. 30, 969, 1984) using a Refletron ® instrument (Boehringer Mannheim). Surprisingly, even with this highly concentrated insulin solution, good biological efficacy (decrease in blood glucose levels after inhaled insulin administration) was found. Fig. 5 illustrates this result.

The aqueous aerosol formulations of the invention may contain, if desired, in addition to the active ingredient and water other solvents - such as ethanol. The amount of ethanol will be limited, depending on the solubility properties of the active substances, so that at too high concentrations the active substance may fall out of the aerosol formulation. Solution stabilizing additives such as pharmacologically acceptable preservatives such as ethanol, phenol, cresol or paraben, pharmacologically acceptable acids, bases or pH adjusting buffers, or surfactants are possible. In addition, metal chelating agents - such as EDTA - may be added to stabilize the solution or improve aerosol quality. To improve the solubility and / or stability of the active ingredient in the aerosol formulation, amino acids such as aspartic acid, glutamic acid (asparagine, glutamine) and in particular proline can be added.

In addition to interferons, superoxide dismutase and insulin, the following active ingredients embody the preferred active ingredients of the medicament preparation of the invention:

antisense oligonucleotides, orexins, erythropoietin, tumomecrotic factor alpha, tumomecrotic factor beta,

G-CSF ("granulocyte colony stimulating factor"), GM-CSF ("granulocyte macrophage colony stimulating factor"), annexin, calcitonin, leptin, parathyroid hormone, parathyroid hormone fragment, interleukins such as interleukin 2, interleukin 10, interleukin 12 "Intercellular adhesion molecule), somatostatin, somatotropin, tPA (" Tissue plasminogen activator), TNK-tPA, tumor-associated antigens (such as peptide, protein or DNA), peptide bradykinin antagonists, produce latin,

GHRH (growth hormone releasing hormone)

CRF (corticotropin releasing factor),

ΕΜΑΡ II, heparin, soluble interleukin receptors such as sIL-1 receptor, vaccines such as hepatitis or measles vaccine, antisense polynucleotides, transcription factors.

Claims (29)

1. A propellant-free nebulizer comprising an aerosol formulation in the form of a solution with water or a water / ethanol mixture as solvent, characterized in that it contains, in a concentration of 25 to 100 mg / ml, an active substance selected from the group consisting of: antisense oligonucleotides, orexins, erythropoietin, tumomecrotic factor alpha, tumorecrotic factor beta, Granulocyte colony stimulating factor - G-CSF granulocyte macrophage colony stimulating factor - GM-CSF annexin, calcitonin, leptin, parathyroid hormone, parathyroid hormone fragment, interleukins such as interleukin 2, interleukin 10, interleukin 12, Intercellular adhesion molecule - soluble ICAM somatostatin, somatotropin, Tissue plasminogen activator - tPA TNK-tPA, tumor-associated antigens, such as peptide, protein or as DNA, peptide bradykinin antagonists, urodilatin, Growth hormone releasing hormone - GHRH Corticotropin releasing factor - CRF ΕΜΑΡ II, heparin, soluble interleukin receptors such as sIL-1 receptor, vaccines such as hepatitis or measles vaccine, antisense polynucleotides, transcription factors, and which is suitable for measuring a therapeutically effective amount of a single dose of aerosol preparation in a metered dose and spraying it under a high pressure range of 10 to 50 MPa, through at least one nozzle with a hydraulic diameter of 1 to 12 microns, to form inhalable drops with an average particle size of less than 10 microns. Propellant-free atomiser according to claim 1, characterized in that the active substance is insulin. Propellant-free nebulizer according to claim 2, characterized in that the active substance is insulin in a concentration of 25 to 60 mg / ml. Propellant-free nebulizer according to claim 2, characterized in that the active ingredient is insulin in a concentration of greater than 30 mg / ml. Propellant-free nebulizer according to claim 2, characterized in that the active ingredient is insulin in a concentration of 30 to 60 mg / ml. Propellant-free nebulizer according to claim 2, characterized in that the active ingredient is insulin in a concentration of 33 to 40 mg / ml. Propellant-free atomiser according to claim 1, characterized in that the active substance is superoxide dismutase. Propellant-free atomiser according to claim 1, characterized in that the active substance is interferon. Propellant-free nebulizer according to claim 1, characterized in that the active substance is Omega interferon. Propellant-free atomiser according to any one of claims 1 to 9, characterized in that the aerosol formulation comprises one or more excipients from the group consisting of surfactants such as surfactants, emulsifiers, stabilizers, permeation enhancers and / or or preservatives. Propellant-free atomiser according to any one of claims 1 to 10, characterized in that the aerosol formulation comprises an amino acid. Propellant-free nebulizer according to claim 11, characterized in that the aerosol formulation comprises proline, aspartic acid or glutamic acid, which are suitable for improving the solubility or stability of the active substance. The propellant-free atomiser according to claim 1, characterized in that the aerosol formulation at 25 ° C has a viscosity of up to 1600 x ^{10 6} Pa s measured with an Oswald viscometer. 14. Propellant-free atomiser according to claim 1, characterized in that the aerosol preparation has a 25 ° C viscosity of 900.10 ^"6 Pa.s. up to 1600 x ^{10 6} Pa.s, measured with an Oswald viscometer. The propellant-free nebulizer according to claim 1, characterized in that the aerosol formulation at 25 ° C has a viscosity of 900.10 ⁶ Pa.s. up to 1100.10 " ⁶ Pa.s., measured with an Oswald viscometer. 16. A propellant-free nebulizer according to claim 1, characterized in that the aerosol formulation has a viscosity at 25 ° C of from 950 to ⁶ Pa.s. to 1300.10 ^-6 Pa, measured with an Oswald viscometer. Propellant-free atomiser according to claim 13 or 15, characterized in that the active substance is superoxide dismutase. Propellant-free atomiser according to claim 13 or 15, characterized in that the active substance is interferon, preferably interferon omega. Propellant-free nebulizer according to claim 13 or 15, characterized in that the active substance is insulin. A propellant-free nebulizer according to any one of claims 1 to 9, characterized in that it is small enough to be worn in a jacket pocket or handbag. A propellant-free nebulizer according to any one of claims 1 to 19, characterized in that it has a nozzle body with two nozzles arranged so that the two jets meet in a manner in which the aerosol formulation is atomized. A method for preparing propellant-free aerosols for administration by inhalation, characterized in that in a propellant-free nebulizer, a therapeutically effective amount of a single dose of the aerosol formulation according to any one of claims 1 to 20 is measured and sprayed under high pressure between 10 MPa and 50 MPa through at least one nozzle with a hydraulic diameter of 1 to 12 microns for inhalable droplets with a mean particle size of less than 10 microns. The method of claim 22, wherein the aerosol formulation is sprayed over a period of 1 to 2 seconds. The method of claim 22 or 23, wherein the effective amount of a single dose is between 10 and 20 microliters. The method of any one of claims 22 to 24, wherein the atomizer has a nozzle body with two nozzles arranged such that the two jets meet in a manner in which the aerosol formulation is atomized. A method according to any one of claims 22 to 25, characterized in that the active ingredient is insulin. The method of claim 26, wherein the insulin is at a concentration of 25 mg / ml to 60 mg / ml, preferably 30 to 40 mg / ml. Use of a propellant-free nebulizer according to any one of claims 1 to 21, comprising an aerosol formulation according to any one of claims 1 to 21, for the preparation of an aerosol with an average particle size of less than 10 μιη. Use according to claim 27, wherein the aerosol formulation used is an aqueous solution containing more than 25 mg / ml insulin, preferably more than 30 mg / ml insulin, more preferably 30 to 60 mg / ml insulin, even more preferably 33 to 40 mg / ml insulin .